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Extremophiles - Thermophiles
The Thermoacidophile - Sulfolobus solfataricus Sulfolobus solfataricus is a crenarchaeon with both highly thermophilic and acidophilic tendencies. It can be found at temperatures ranging from 50-87 degrees Celsius. However, optimal growth is observed at 85 degrees Celsius. Additionally, this organism perfers acidic environments ranging from pH 2-4.5. It has also been noted that Sulfolobus solfataricus is a obligate anaerobe requiring oxygen for survival. It has also been given the status of a facultative autotroph as it can metabolize sulfur. Sulfolobus solfataricus are irregularly shaped. (see Figures 1 and 2) They also tend to be 0.8 to 1.0 um in diameter. As observed with other archaean the cell wall lacks peptidoglycan. The wall also lacks hexosamine, but has a high level of carbohydrates within the cell wall. Early discovery of Sulfolobus included isolations from both aquatic and terrestrial locations at Yellowstone, Italy, Dominica, and El Salvador. Later publications also claimed to isolate the organism in Lassen National Park(California), Kamchatka (Russia), and Iceland. Despite the widespread distance of these habitats, the Sulfolobus Solfataricus found in each only vary slightly in DNA sequences. This variation is typically in terms of the distances between protein coding regions. 'Background ' Sulfolobus was discovered in 1972 through a collaborative effort between two labs one based in Indiana and one in Wisconsin. (1) They initially classified sulfolobus as a bacteria, later Sulfolobus was classified as a member of archaea. The early isolations from the genus Sulfolobus were from thermal acidic areas, both aquatic and terrestial. The archaean was found in the hotsprings of Yellowstone National Park, El Salvador, Dominica, and Italy where virtually all the temperatures ranged from 65-90 degrees Celsius and had a pH between 1.5 and 2.4. (1) Within their native habitats Sulfolobus is abundant, making it easy for researchers to visualize using direct phase microscopy of soil and water samples. (1) Cultures containing Sulfolobus were obtained from their respective environment and then enriched in one of two mediums: a 0.1% yeast extract or on elemental sulfur.(1) The cultures were able to grow under both sets of circumstances suggesting Sulfolobus was a facultative autotroph. Sulfolobus morphology was retained in the lab cultures. (1) Following experimentation, a sample representing a species of Sulfolobus was given to the American Type Culture Collection. 'Sequencing the Genome ' Sulfolobus solfataricus was the first Archaea to have its genome sequenced. The sequence of Sulfolobus solfataricus was also the first complete genome project undertaken by Canada. (2) Laboratories in the US, Denmark, France, and the Netherlands partnered with the Canadian labs one year following the start of this project to complete the genome sequence in 2001. (2,3) The first step in sequencing the genome was to isolate the genomic DNA from the Sulfolobus solfataricus cells. The genome was then partially digested with the restriction enzymes BamHI or HindIII. The resulting fragments were on average 40kbp long and were incorporated into a cosmid vector: Tropist 3. Later partially digested fragments were also incorporated into lambda and bacterial artificial chromosome libraries. These libraries were sequenced and some regions were checked with PCR from both ends. Around 30,000 reads were done for the complete sequence lending to the overall average coverage to be over 5 fold. The Genome The initial estimates of the G+C content in the genome were around 60%, however further investigation showed that the genome was only about 36% G+C. The genome is composed of one chromosome with 2,992,245 base pairs. 3,032 genes were identified within the genome. Within the genes encoded, there are 2,977 proteins as well as many RNAs. Interestingly one third of the proteins encoded have no homologs in other sequenced genomes (as of Feb. 2001) . Forty percent of the proteins encoded appear to be archaeal specific, 12% are shared with bacteria, and 2.3% are shared with eukarya. It has been estimated that 11% of the genome contains mobile elements. There were 52 gene families identified within the sequnce. The largest family is involved in fatty acid biosynthesis - with a higher concentration of acetyl-CoA synthetases dehydrogenases (17 members), ATP binding subunits of ABC transporters (19 members) There are about 420 copies of large cluster tandem repeats (LCTR) within the genome. Where one LCTR is composed of 1 repeat of 20 nucleotides occuring either 3 or 4 times. Functional Genes within the Genome. The Sulfolobus solfataricus genome has a variety of functional genes some of which are unique and others that resemble genes in other organisms. Below we will take a closer look at what some of these genes are and what they are involved in. DNA binding Proteins Two putative DNA binding protein families exist within the genome. These proteins are small, basic, and refenced as Sso7d and Sso10d. There are three Sso7d proteins about 7kDa, that only bind to the minor groove of DNA. This binding results in sharp kinks within the DNA structure. Orthologs to this protein only exist in the genus Sulfolobus. Sso10d is a 10kDa protein, that is hypothesized to be involved in DNA replication and repair. This protein is only found in archaea. A chromosome structural maintenance protein Sso2249 also exists, belonging to a family of cohesins/condensins. This protein shares 46-48% of of its sequence with other similar archaeal proteins. While no proteins in this organismn share an ancestry with eukaryote histones, a protein was predicted (with two paralogs- Sso0009, Sso1117, Sso0028 respectively) that belongs to a family of histone deacetylases common to archaea and eukarya. DNA within the Cell Cycle Sulfolobus solfataricus P2 regulates the cell cycle using a CDC6 homologs (Sso0257, Sso0771, and Sso2184). CDC6 in eukaryotes is a regulator of DNA replication. CDC6 is not observed throughout archaea - only in some such as Sulfolobus solfataricus and Halobacterium. The hypothesized origin of replication lies at Sso0771, due to its high GC content and codon skew analysis. It is also possible that more ORC exist, however the genome exists in a complex form not permitting a full evaluation of the number and location of other ORC (?). Three DNA polymerases are thought to be encoded within the genome: B1 (Sso0552), B2 (Sso1459), B3 (Sso0081), with B1 and B3 containing all the exonuclease and polymerase structural motifs. Primases are also found within the chromosome (Sso0079, Sso1048). One ATP-dependent DNA ligase was identified (Sso0189). Four topoisomerases were found with two being ATP dependent reverse gyrases and one being ATP independent type I. DNA Repair and Recombination Within the genome, several repair proteins have been observed. One group contributes to reversal of lesion systems (meaning they repair damage in the DNA structure - a base or in somecases the phosphate-sugar backbone). This group includes the protein phr (photoreactivation enzyme) and other cysteine-S-methyltransferases. Endonucleases involved in repair include endonuclease III, IV, and V and a homolog of Rad2/Fen-1. Two mutT genes were also observed (enzymes that detoxify the nucleotide pool). As a means to repair DNA, sometimes an organims will use recombination. Recombination related proteins that were observed were Xer, 2 RadA proteins, Hjc and Hje. Additionally about 15 helicases potentially involved in DNA repair and recombination have been observed within the chromosome as well as a putatitve bypass polymerase (Sso2448).. The system does not include uvrABC system found in other archaea and does not include mutS/L mismatch repair genes. 'Transcription' It has generally been observed, that transcription in archaea is very similar to eukarya. TATA-binding protein (TBP) and a homolog to transcription factor IIB (TFB) were found within the genome. (Both proteins help initiate transcription.) Archaea all have TFIIE alpha subunit - which is also present in this organism. (Eukarya genomes include TFIIA, TFIIEB, TFIIF, and TFIIH, but these are all absent from Sulfolobus sulfataricus.) In eukarya, RNA polymerases have an integral role in transcription, in that they create an mRNA complementary to the DNA in an organism's genome. Sulfolobus sulfataricus also have RNA polymerases - 14 RNA polymerase subunits are present encoding rpoDN, rpoHB-"B"AA", rpoG, rpoL, rpoE'E", rpoF, rpoK, rpoP. Ribosomal factors are also present as well as transcription elongation factors. 'Translation' Translational aspects include both archaeal-specific features as well as similarities to bacteria and eukarya. Shine-Dalgarno sequences are present, however they are not incorporated into the genome in a traditional manner. There are 46 tRNA's encoded within the genome, only 43 of these tRNA's have different anti-codons. tRNA synthetases are included for every amino acid except for asparagine and glutamine - which are thought to require amidotransferases for their synthesis, especially as multiple subunits for Glutamine-tRNA amidotransferase were observed. The rRNA appears to be present in the 16S and 23S form. These subunits are not cotranscribed with the tRNAs or 5S or 7S RNA. Other proteins that can modify tRNA and rRNA include archaeal intron splicing endonuclease, N-6 methylase, and tRNA nucleotidyl transferase. There is also a total of 65 ribosomal proteins encoded. Chaperonins are also incorporated into the genome: thermosome chaperonin TF55 with all three subunits(alpha, beta, and gamma). Other archaea only include alpha and beta subunits. 'Growth and Metabolism' The cytosol of Sulfolobus solfataricus is at pH 6.5 (in contrast to the environment at pH 2-4). The discrepancies in pH facilitate a pH gradient and indirectly a proton motive force. To moderate the difference in pH, the cell generates a positive charge within the cell using potassium transporters like Trk-like potassium transporter (Sso1757). The unusual charge distributionthat enables ATP production through ATP synthase subunits (eight). The proton . The proton gradient is also responsible for the intake of fuel in the form of inorganic and organic solutes through a secondary transport system. Fuel is also obtained through the ABC transport system - transporters embedded in the membrane. Peptide degradation is via extracellular and intracellular proteasome subunits. (Sso2045, Sso1141, Sso2551 and Sso0277, Sso0738, Sso2675) Carbohydrate degradation is enabled through extracellular cellulases. There are also enzymes that break down glycogen and trehalose including glycosyl hydrolases such as B-glycosidase, a-fucosidase, B-xylosidase, a-xylosidase, and B-glucuronidase. 'Transposable Elements' Transposable Insertion elements compose approximately 10% of the Sulfolobus solfataricus genome. Two hundred complete transposable insertion elements have been observed in the genome of Sulfolobus solfataricus. 184 of these elements can be associated with seven known bacteria/eukarya families. Association with the remaining elements are unknown. Some have hypothesized that they are linked to eukarya, but it is not known. 44 additional partial copies of insertion elements are also present. Several IS elements appear in the genome suggesting a duplication may have occurred. Sources: 1. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Brock TD, Brock KM, Belly RT, Weiss RL. Arch Mikrobiol. 1972;84(1):54-68. 2. Completing the sequence of the Sulfolobus solfataricus P2 genome. 3. The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Qunxin She, Rama Singh, Fabrice Confalonieri, Yvan Zivanovic, Ghislaine Allard, Mariana Awayez, Christina Chan-Weiher, Ib Groth Clausen, Bruce Curtis, Anick De Moors, Gael Erauso, Cynthia Fletcher, Paul Gordon, Ineke Heikamp-de Jong, Alex Jeffries, Catherine J. Kozera, Nadine Medina, Xu Peng, Hoa Phan Thi-Ngoc, Peter Redder, Margaret Schenk, Cynthia Theriault, Niels Tolstrup, Robert Charlebois, W. Ford Doolittle, Michel Duguet, terry Gaasterland, Roger Garrett, Mark Ragan, Christoph Sensen, John Van der Oost. Proc Natl Acad Sci U S A. 2001 July 3; 98(14): 7835–7840. 4. Biogeography for Bacteria. Tom Fenchel. Science, Microbiology. July 24 2003 5. Sulfolobus: A New Genus of Sulfur-Oxidizing Bacteria Living at Low pH and High Temperature. Thomas Brock, Katherine Brock, Robert Belly, and Richard Weiss. Arch. Mikrobiol. 84, 54--68 (1972) .